This invention relates broadly to a class of oral vaccines for the prevention of enteric disease. A living, non-pathogenic mutant, oral vaccine strain of Salmonella typhi has already been shown to be safe and effective in protecting against typhoid fever; it is a mutant, galactose epimeraseless strain of S. typhi designated as Ty21a. Its preparation, safety, and efficacy as an oral vaccine have already been described in Germanier, R. and Furer, J. Infect. Dis. 131:553-558, 1975; Wahdan, N. H. et al., Bull. WHO. 58:469-474; and U.S. Pat. No. 3,856,935 to R. Germanier, the disclosure of which is hereby incorporated by reference.
Bacterial diseases of the gastrointestinal tract usually occur by one of three overall mechanisms. The first mechanism, termed "intoxication," occurs by bacterial secretion of an exotoxin that oftentimes is preformed in food prior to ingestion by the host. This process is exemplified by staphylococcal or clostridial food poisoning. In contrast, the remaining two processes require living and multiplying disease agents. In the "enterotoxigenic" mechanism, bacteria colonize the small intestine, usually in the jejunum or duodenum. These bacteria multiply on the intestinal surface and elaborate an enterotoxin that stimulates excessive fluid and electrolyte efflux resulting in a watery diarrhea. Enterotoxigenic Escherichia coli and Vibrio cholera serve as typical examples. Finally, a third group of organisms, termed "invasive," actually penetrate the epithelial mucosa of the large intestine. Subsequently, these organisms multiply intracellularly and disseminate within or through the mucosa. This latter mechanism, classically typified by Shigella and Salmonella, is now thought to be used by invasive strains of E. coli, Yersinia, and, possibly, Campylobacter. In contrast to other invasive bacterial diseases like salmonellosis, in which the invading bacteria are disseminated throughout the host, shigellosis is a disease normally confined to the intestinal lining. Thus, these features distinguish the toxigenic from the invasive mechanism of intestinal disease.
Two common and essential features of invasive bacteria are their ability to penetrate and to multiply within the epithelial cells of the colon. Mutants of Shigella strains that fail to penetrate or that penetrate but cannot multiply intracellularly have been isolated. Both types of mutants are avirulent. The process of invasion has thus far been characterized in microscopic, but not biochemical detail. The first visible alteration in the host intestinal epithelium is a localized destruction of the microvilli, the outermost structure of the intestinal lining. The invading bacteria are then engulfed by means of an invagination of the intestinal cell membrane and are contained intracellularly within vacuoles. Subsequently, the microvilli are reestablished and intracellular bacterial multiplication occurs. These bacterial then destroy the vacuole and disseminate to adjacent cells, causing necrosis and resulting in acute inflammation and focal ulceration of the epithelium. The resulting dysentery is characterized by a painful, bloody, and mucous diarrhea, normally of relatively small volume.
Genetic studies of Shigella flexneri have previously resulted in the conclusion that virulence is multideterminant, with at least two widely separated bacterial chromosomal regions being required for invasion. Furthermore, these studies have shown that not only is a smooth lipopolysaccharide bacterial cell surface necessary for intestinal invasion, but also that only certain O-repeat unit polymers are effective in this process; this is true for both shigellae and invasive E. coli. Until recently, plasmids did not appear to play a role in the invasion process or in the virulence of Shigella. Recent evidence amassed over the past three years, however, demonstrates that plasmids of Shigella are involved in the invasion process.
Bacillary dysentery remains highly endemic in many areas of the world and still is a significant cause of illness in developed countries. There are over thirty serotypes of the organisms which cause shigellosis (bacillary dysentery), the prominent members of which are S. sonnei, S. flexneri, S. dysenteriae and S. boydii. There are six serologically separable S. flexneri types (i.e. I through VI) of which S. flexneri types IIa and III are responsible for the majority of S. flexneri infections. In the United States and northern Europe, S. sonnei is responsible for more than 65 percent of the cases. Together with S. flexneri IIa and III strains, S. sonnei strains cause greater than 90% of all shigellosis worldwide. Parenteral vaccines have not been effective in protecting against bacillary dysentery because shigellosis is an infection limited to the superficial layer of the colonic mucosa. It is, therefore, not surprising that attempts to immunize man or other primates with killed vaccines or even living virulent organisms, administered by the parenteral route, have not been successful.
Living, attenuated, oral Shigella strain vaccines have been demonstrated to be protective against bacillary dysentery under both laboratory and field conditions. It has been suggested that the local intestinal immune response which is induced by the living oral vaccines inhibits invasion of intestinal epithelial cells by the pathogen. This immunity has been associated with the type-specific somatic antigen of the vaccine strain. None have come into widespread use because of difficulties in isolating safe, genetically-stable (non-reverting) strains or because of the large number of doses required to produce immunity.
S. sonnei produce a characteristic cell surface antigen, termed form I, which has altruonic acid as a component of its O-specific side chain. Recently, it has been demonstrated that the form I antigen is encoded by a large non-conjugative plasmid (Kopecko, D. J. et al., Infect. Immun. 29:207-214, 1980). This fundamental study found that by utilizing a plasmid mobilizing system, transfer of the form I antigen synthesizing genes was possible to certain specific Shigella flexneri and Salmonella typhi strains, or retransfer to a form II S. sonnei strain was possible.
Unlike shigellae, the typhoid bacillus causes a systemic infection following penetration of the intestinal mucosa, and parenteral vaccines have been shown to be effective against this infection. These parenteral vaccines do, however, elicit significant side effects which include fever, malaise, headache, and localized reactions at the site of inoculation. Since the safety (i.e., freedom from the above-mentioned side effects) and immunogenicity of S. typhistrain Ty21a has been established, it was considered that this attenuated strain might be utilized as a carrier organism for other protective antigens and, thus, could be used as an oral vaccine to protect against an enteric infection other than typhoid fever or simultaneously against both typhoid fever and also other enteric infections. By protective antigen we mean a molecular structure, either somatic or soluble, which stimulates production of one or more antibodies and protects against a specific enteric disease. Additionally, the term "carries", "carried" or "carrier" is not to be construed as limiting the invention to a specific derivative or method of modification of the parent galE S. typhi strain. The term does not imply that the genetic determinant necessarily has to be plasmid-borne; conversely, it can be part of the bacterial chromosome.